An olfactory receptor influences melanocyte pigmentation

1

1 Functional Characterization of the Odorant Receptor 51E2 in Human Melanocytes*

Lian Gelis‡,1,2

, Nikolina Jovancevic‡,1

, Sophie Veitinger‡, Bhubaneswar Mandal

§,¥, Hans-Dieter

Arndt§

, Eva M. Neuhaus†,1

and Hanns Hatt‡,1

‡Cell Physiology, Ruhr-University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany

§Organic Chemistry I, Friedrich Schiller University, Humboldtstr. 10, 07743 Jena, Germany

†Pharmacology and Toxicology, University Hospital Jena, Drackendorfer Str. 1, 07747 Jena, Germany

¥New Address: Department of Chemistry, IIT Guwahati, North Guwahati, Assam - 781 039, India

1Both authors contributed equally.

2To whom correspondence should be addressed: Cell Physiology, Ruhr-University Bochum,

Universitaetsstrasse 150, 44801 Bochum, Germany, lian.gelis@rub.de, tel +49-234-3224586

*Running title: An olfactory receptor influences melanocyte pigmentation

Key words: PSGR; GPCR; pigment; cell signaling; melanocyte; calcium imaging; cell differentiation;

cell proliferation

ABSTRACT

Olfactory receptors, which belong to the

family of G-Protein coupled receptors, are

found to be ectopically expressed in non-

sensory tissues mediating a variety of cellular

functions. In this study, we detected the

olfactory receptor OR51E2 at the transcript

and protein level in human epidermal

melanocytes. Stimulation of primary

melanocytes with the OR51E2 ligand -ionone

significantly inhibited melanocyte proliferation.

Our results further showed that -ionone

stimulates melanogenesis and dendritogenesis.

Using RNA silencing and receptor antagonists

we demonstrated that OR51E2 activation

elevated cytosolic [Ca2+

] and [cAMP], which

could mediate the observed increase in melanin

synthesis. Co-immunocytochemical stainings

using a specific OR51E2 antibody revealed

subcellular localization of the receptor in early

endosomes associated with EEA-1. Plasma

membrane preparations revealed that OR51E2

protein is present at the melanocyte cell surface.

Our findings thus suggest that activation of

olfactory receptor signaling by external

compounds can influence melanocyte

homeostasis.

Olfactory receptor (OR) genes constitute the

basis for the sense of smell and were first

described as being expressed exclusively in the

olfactory epithelium (1). Later on, a subset of

human OR genes have been found to be expressed

in various non-olfactory tissues (2-4). This ectopic

expression of OR genes raised the possibility that a

subset of ORs may have physiological functions in

non-olfactory tissues, in addition to their canonical

role in odor detection in the sensory neurons.

Functionality of olfactory receptors in non-

olfactory cell types is becoming better understood.

Initially, a lack of correlation in expression levels

in different tissues between human-mouse

orthologous pairs and between intact and

pseudogenized ORs was taken as indication that

functional interpretation of ectopic OR expression

cannot be based on transcriptional information (5).

Analysis of non-olfactory tissues in human and

chimpanzee later revealed that OR orthologous

genes are expressed in the same tissues more often

than expected by chance alone. Orthologous OR

genes with conserved ectopic expression have

http://www.jbc.org/cgi/doi/10.1074/jbc.M116.734517The latest version is at JBC Papers in Press. Published on May 18, 2016 as Manuscript M116.734517

Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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An olfactory receptor influences melanocyte pigmentation

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evolved under stronger evolutionary constraints

than OR genes expressed exclusively in the

olfactory epithelium (6), indicating that ectopically

expressed OR genes have conserved functions in

non-olfactory tissues. In the studies performed so

far, functional analysis of ectopically expressed

ORs revealed a possible role in sperm chemotaxis

(7-9). Human ORs were also shown to mediate

serotonin release in enterochromaffin cells (10),

and to influence proliferation of prostate cancer

cells (11,12). The OR2A4 that localizes to the

cytokinetic structures in cells was shown to

participate in cytokinesis (13). The mouse OR

MOR23 exerts a role in skeletal muscle

regeneration by regulating cell adhesion and

migration (14). In the murine kidney, ORs and

olfactory signaling proteins control the glomerular

filtration rate, renin secretion and blood pressure

(15,16). A synthetic sandalwood compound was

shown to accelerate wound healing processes by

activation of OR2AT4 expressed in human

keratinocytes (17).

In contrast to olfactory neurons, where

activation of the receptors elicits a receptor

current, activation of ectopically expressed

receptors can have diverse effects. ORs are G-

protein coupled receptors (GPCRs), which can

couple to different intracellular signaling cascades

depending on the activation of different types of

heterotrimeric G-proteins (18), the interaction with

other cellular partners such as arrestins (19) and

scaffolding proteins (20), (hetero-)dimerization

with other receptors (21), lipid-protein interactions

(22), and on the intracellular localization (23).

GPCRs, which are activated by chemical ligands

such as small amines, peptide hormones,

chemokines, and odorants, can elicit a huge variety

of cellular responses, among them cell shape

changes and altered adhesion (24), as well as

changes in cell proliferation (25).

In the present study, we functionally

characterized the signaling pathway and the

implication of activation of OR51E2 in human

melanocytes. We found, that OR activation in

these cells elicits Ca2+ signals, and ultimately

regulates cellular proliferation and differentiation,

similar as observed previously in prostate

epithelial cells (11).

EXPERIMENTAL PROCEDURES

Cell culture and transfection- Reagents for

cell culture use were purchased from Life

Technologies (Carlsbad, CA, USA), unless stated

otherwise. Hana3a cells were maintained under

standard conditions in DMEM supplemented with

10% FBS, 100 units/ml penicillin and

streptomycin, and 2 mM L-glutamine. Hana3a

cells were transiently transfected (3 μg of OR

cDNA per dish) in 35 mm dishes (Falcon, BD

Bioscience, Heidelberg, D) using a standard

calcium phosphate precipitation technique.

Primary neonatal normal human epidermal

melanocytes (NHEM) were a generous gift from

Dr. G. Neufang (Beiersdorf, Hamburg, D).

Melanocytes were maintained under standard

conditions in melanocyte growth medium (Cell

Systems, St. Katharinen, D). For siRNA

experiments, melanocytes were transiently

transfected with either targeted or scrambled

siRNAs using Lipofectamine 2000 (Life

Technologies) according to manufacturers’

instructions. The transfection rates were less than

1% with both OR51E2 siRNA and control siRNA

as detected by co-expression of GFP. Cells were

analyzed in Ca2+ imaging experiments two days

after transfection

Antibodies- The following primary antibodies

were used: (a) rabbit polyclonal antibody against

OR51E2 (Eurogentec, Seraing, BE); (b) mouse

polyclonal antibody against human EEA-1

(Abcam); (c) rabbit polyclonal antibodies against

human p44/42 MAPK and against human p38

MAPK (Cell Signaling, Danvers, MA, USA); (d)

rabbit polyclonal antibodies against human

phosphorylated p44/42 MAPK and against human

phosphorylated p38 MAPK (Cell Signaling); (e)

mouse monoclonal antibody against

glycerinaldehyde-3-phosphate-dehydrogenase

(GAPDH) (Abcam); (f) mouse monoclonal

antibody against human tyrosinase (Santa Cruz

Biotechnology, Dallas, TX, USA); (g) mouse

monoclonal antibody against rhodopsin, 4D2

(Abcam); (h) rabbit monoclonal against

proliferating cell nuclear antigen (PCNA; Abcam)

(i) mouse monoclonal alkaline phosphatase-

coupled antibody against digoxin (Sigma); (j)

secondary goat-anti-rabbit and goat-anti-mouse

antibodies conjugated to HRP (Bio-Rad

Laboratories, Hercules, CA, USA) and (k)

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secondary goat-anti-rabbit and goat-anti-mouse

antibodies conjugated to Alexa Fluor 546 or Alexa

Fluor 488 (Life Technologies).

Western Blotting- 80% confluent cells were

harvested by scraping and proteins were prepared

by standard methods. Sample aliquots of

fractionated melanocytes were mixed with

Laemmli buffer (30% glycerol, 3% SDS, 125 mM

Tris/Cl, pH 6.8), resolved by 10% SDS-PAGE,

and transferred to nitrocellulose membrane

(Protan, Schleicher & Schuell, Dassel, DE). The

nitrocellulose membranes were stained with

Ponceau S (Sigma, Munich, DE), blocked with

TBST (150 mM NaCl, 50 mM Tris-Cl, Tween 20,

pH 7.4) containing 5% non-fat dried milk (Bio-

Rad), and incubated with the primary antibody

diluted in 3% dry milk in TBST. After washing

and incubation with horseradish peroxidase-

coupled secondary antibodies, detection was

performed with ECL plus (Amersham,

Braunschweig, DE) and the Fusion-SL image

acquisition system (Vilber Lourmat Deutschland

GmbH, Eberhardzell, DE). Signal intensities were

quantified using ImageJ (v1.4.3.67, Broken

Symmetry Software).

Cell Surface Protein Isolation- Biotinylation

and isolation of cell surface proteins for Western

Blot analysis were preformed using the Thermo

Scientific™ Pierce™ Cell Surface Protein

Isolation Kit according to the manufacturer’s

instructions (Thermo Fisher Scientific, Pierce,

Rockford, IL, USA).

Single cell Ca2+ imaging- Melanocytes were

incubated for 45 min and Hana3a cells for 30 min

in loading buffer (pH 7.4) containing Ringers’

solution and 7.5 µM Fura-2-AM (Life

Technologies). After removal of extracellular

Fura-2 by washing, ratiofluorometric Ca2+ imaging

was performed using an inverted microscope

equipped for ratiometric live cell imaging (IX71,

Olympus, Hamburg, D) with a 150-watt xenon arc

lamp, a motorized fast change filter wheel

illumination system for multiwavelength excitation

(MT20, Olympus), and a 12-bit 1376 × 1032-pixel

charge-coupled device (CCD) camera (F-View II,

Olympus). WinNT-based cellRTM imaging

software (Olympus) served to collect and quantify

spatiotemporal Ca2+-dependent fluorescence

signals (excited at 340 and 380 nm, measured at

510 nm, and calculated as a ƒ340/ƒ380 intensity

ratio) of the cells. Compounds were diluted in

Ringer’s solution or for Ca2+free experiments in

Ringer’s solution containing 50 µM EGTA and

applied using a specialized pressure-driven

microcapillary perfusion system designed for

instantaneous solution change and focal

application. Images were acquired in randomly

selected fields of view. Ultrapure α-ionone and β-

ionone were a generous gift of Dr. J. Panten

(Symrise, Holzminden, D). Steroids were

purchased from Steraloids, Newport, RI),

aminoethoxydiphenyl borate (2-APB), SKF

96365, GdCl3 and BTP2 from Tocris (Tocris

Bioscience, Bristol, UK), Endothelin-1,

Thapsigarigin and ATP from Sigma. Odorants

were prediluted in DMSO (Sigma), so that the

DMSO concentration did not exceed 1‰ (v/v),

which was well tolerated by melanocytes. All

odorants assayed for potential inhibition of

OR51E2 were tested in at least three different

series of transfection experiments.

Immunocytochemistry- Cells were seeded on

coverslips and maintained as described above. The

cells were fixed by incubation with 4%

paraformaldehyde at 4°C for 30 min. After

blocking with 1% gelatin for 1 h, cells were

incubated overnight with the primary antibody.

For visualization, secondary fluorescent IgGs (Life

Technologies) (1:1000) were used. Micrographs

were taken by using a LSM510 Meta confocal

microscope (Zeiss, Jena, D) Apoptosis detection- 50-70% confluent

melanocytes were stimulated for 72 h with

different concentrations of β-ionone or solvent

only. Apoptotic activity was detected by Caspase-

Glo® 3/7 Assay (Promega, Madison, WI).

Apoptotic cells were quantified using the terminal

deoxynucleotidyl transferase dUTP nick end

labeling (TUNEL) based In Situ Cell Death

Detection Kit (Hoffmann-La Roche, Basel, CH)

according to the manufacturer’s instructions.

Cell Proliferation- Growing melanocytes

were plated in 96 well plates at a density of 5×103

cells/well. After 24 hours at 37 °C with 5% CO2,

cells were treated with different concentrations of

-ionone. Cell proliferation was assessed after 6

days using CyQUANT cell proliferation assay kit

(Life Technologies). For the visualization of

proliferating cells via PCNA staining, cells were

stimulated for 6 days with β-ionone (50 µM) or

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solvent only. Afterwards, cells were stained with

anti-PCNA antibody (1:500) as described in

section ‘Immunocytochemistry’ and with Alexa

Fluor® 546 Phalloidin (Life Technologies; 1:200).

DNA and siRNA constructs- PSGR targeted

and scrambled hairpin siRNA designs were carried

out with siRNA Target Designer-Version 1.51

(Promega, Madison, WI) as described previously

(11). The best working siRNA sequence of PSGR

was gctgcctcctgtcatcaat; the oligonucleotide

sequences to generate 5'-target-loop-reverse-

complement-3' hairpins were:

5’tctcgtgcctctgtcatcaataagttctctattcatcacaggaggcag-

cct-3’, 5’-ctgcaggctgcctcctgtcatcaatagagaacttattc-

atgacaggaggcagc-3’.

The following scrambled versions of the siRNA

sequence were used as control:

5’-tctcgtacactgaccccctttgtaagttctctacaaagggggtca-

gtgtacct-3’, 5’-ctgcaggtacactgacccccttctagagaac-

ttacaaagggggtcagtgtac-3’.

RT-PCR- RNA of melanocytes was isolated

with the RNeasy Mini Kit including on-column

DNase digestion (Qiagen, Hilden, D) according to

manufacturers’ instruction. RNA concentration

and quality (A260/A280 ratio) were analyzed

using Spectrophotometer NanoDrop ND-1000

(Thermo Scientific, Waltham, MA, USA). cDNA

synthesis and semiquantitative RT-PCR cDNA

synthesis was performed by using the iScript

cDNA Synthesis Kit (Bio-Rad). Control reactions

with no reverse transcriptase (-RT) served to

exclude contamination with genomic DNA. RT-

PCR was performed using GoTaq qPCR Master

Mix (Promega, Madison, WI, USA) with 2ng of

template cDNA and 10 pmol specific primers

designed for OR amplification. The amplifications

were done for 40 cycles (45°s, 94 °C; 45 s, 60 °C;

45 s, 72 °C). Primers used for RT-PCR were:

Actin 5’-tcttccagccttccttcctg-3’ and 5’-cacggagtac-

ttgcgctcag-3’; Tyr-P1 5’-ggcccgggctcaattcccaa-3’

and 5’-gcaagggccagacctcccga-3’; OR1J2 5’gaacac-

catcccccatgtct-3’ and 5’-tggatccctttggttgaagg-3’;

OR2D2 5’-agtgcgcccttcttgcagtga-3’ and 5’-actg-

cctcggtagggtagcct-3’; OR4N5 5’-ctgcacttgcctttctg-

tgg-3’ and 5’-atatgggtggtgcatgtgga-3’; OR5C1 5’-

ctatgtcgcagcgtctatgc-3’ and 5’-gcagtggagggatatcg-

cag-3’; OR6K3 5’-gaggggatcttgctgaccac-3’ and 5’-

aggcttagcacagggaccaa-3’; OR6T1 5’ ttccccagtagc-

cacctcat-3’ and 5’-caagcatcttgggaaccaca-3’;

OR9G1 5’-ctggctgcctgtgtcagttc-3’ and 5’-accagca-

atgcacacagctt-3’; OR10A4 5’-agcttctcagccctgtc-

cac-3’ and 5’-gggcacaatgaccaagttga-3’; OR10H5

5’-gctcactgtcatgggctacg-3’ and 5’-ccaccaccagcaca-

tcatct-3’; OR11H4 5’-gacccatgaacaggtcagca-3’

and 5’-aggcaaaatttcccagcaga-3’; OR51E2 5’-actg-

ccttccaagtcagagc-3’ and 5’-cttgcctcccacagcctg-3’;

OR2A4/7 5’-ggtgccccggatgctggtg-3’ and 5’-ggggt-

ggcagatggccacg-3’; OR51B5 5’-caatggcaccctcctt-

cttc-3’ and 5’-caagcagaatgccagactcg-3’; OR5P3

5’-tggtagagtttactcttttggggt-3’ and 5’-tgagcatgaca-

ggtgtgact-3’; OR6B3 5’-cagctccctggtgtgcaccg-3’

and 5’-caccagctggaggcacagcc-3’; OR10AD1 5’-

actgcctggttctttgggctga-3’ and 5’-gccaatcactatgg-

gggcctca-3’; OR51E2 (intron-overspanning) 5’-

cctcagccttctgagtcagc-3’ and 5’-gagactgtgacaa-

gccctgg-3’.

Semiquantitative real-time PCR was

performed using GoTaq® qPCR Master Mix

(Promega) with the Mastercycler Realplex2

(Eppendorf, Hamburg, D) (20 µl total volume; 40

cycles of 95 °C, 60 °C and 72 °C at 45 s each). All

experiments were conducted in triplicate. PCR was

performed with cDNA (equivalent of 100 ng total

RNA) and specific primer pairs for the

amplification of tyrosinase and GAPDH. The

housekeeping gene GAPDH was used for relative

quantification by the ΔCt (cycle threshold)

method. Results are reported as fold changes in

gene expression compared to control as calculated

by the 2-ΔΔCt method. Following primer pairs were

used:

Tyrosinase (1) 5’-ggctgttttgtactgcctgc-3’ and 5’-

aggaacctctgcctgaaagc-3’; Tyrosinase (2) 5’-cttcac-

aggggtggatgacc-3’ and 5’-tctctgtgcagtttggtccc-3’;

GAPDH 5’-accacagtccatgccatcac-3’ and 5’-

tcccaccaccctgttgctgta-3’.

cAMP assay- cAMP concentration was

analyzed using a cAMP kit from R&D systems

(Minneapolis, MI). In brief, 7x104 melanocytes

were stimulated with growth medium containing

odorant or forskolin and 100 µM 3-isobutyl-1-

methylxanthin (IBMX) as cAMP-PDE inhibitor.

Afterwards, the cells were lysed in 0.1 M of HCl

in ice-cold ethanol. After 30 min incubation on ice,

cells lysates were desiccated for five hours at

37°C. Samples were reconstituted in 400 µl of

0.1 M HCl. Sample volumes of 100 µl per well

were transferred to a microtiter plate and cAMP

concentrations per well were measured according

to the manufacturers’ instructions with the

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following minor changes: To exclude differences

in anti-cAMP antibody binding efficiency, all

samples and standards were adjusted to identical

ionic conditions, i.e. standards provided by the

manufacturer in 10 mM HCl / ethanol were

desiccated and reconstituted in 0.1 M HCl. For

each tested condition and standard, three replicate

experiments were performed and results were

averaged.

Melanin content assay- Melanocytes were

cultured for 72 h in basal medium containing β-

ionone (50 µM), forskolin (20 µM), α-ionone (200

µM), H89 (10 µM) or the solvent only (0.1 %

DMSO). Melanin contents of stimulated

melanocytes were measured according to the

method of Oka et al. (26) with a slight

modification. After stimulation, cells were

harvested by scraping and cell numbers were

counted using a counting chamber (Blaubrand

Neubauer improved, Sigma). To take the anti-

proliferative effect of β-ionone into account, cell

numbers were adjusted to 1 x 105 before

determination of the melanin content. Cell pellets

were solubilized in boiling 1 M NaOH for 10 min.

Spectrophotometric analysis of melanin content

was performed at 400 nm absorbance.

Differentiation assay- Melanocytes were

cultured for 6 days in basal medium containing β-

ionone (50 µM), forskolin (20 µM) or the solvent

only (0.1 % DMSO). Cell morphology was

checked by bright field microscopy using a Zeiss

Axioskop2 microscope with 20 x magnification.

Undifferentiated (bipolar morphology, small cell

bodies, less pigmentation) and differentiated

melanocytes (multiple dendrited, large cell body,

high pigmentation) were quantified.

Synthesis of FITC-labeled steroids- FITC-

labeled steroids were obtained by chemical

synthesis from dihydrotestosterone and

dehydrotestosterone. In brief, the 17--OH group

of the respective steroid was activated by acylation

with carbonyldiimidazole (27, 28, 29) in

tetrahydrofuran. The resulting monoimidazolide

was treated with excess 4,7,10-trioxa-1,13-

tridecanediamine in actetonitrile, followed by

evaporation and washing with aqueous NaHCO3.

Treatment of the resulting primary amine

intermediate with fluoresceine-5isothiocyanate in

DMF in the presence of Hünig’s base (iPr2NEt)

provided the fluorescently labeled steroid ligands.

The final products were purified to homogeneity

by using preparative HPLC and obtained as orange

powders after lyophilisation in 12-50% overall

yield. Details of synthesis procedures and charac-

terization data for all new compounds (m.p.,

HRMS, 1H- and 13C-NMR, IR) were described in

the following section.

All solvents, when not purchased in suitable

purity or dryness, were distilled using standard

methods. Alternatively, solvents (HPLC grade)

were passed through activated alumina columns

under dry argon atmosphere (solvent purification

system, M. Braun Inertgas-Systeme GmbH,

Garching, Germany). Deionized water was used

for all experiments. All reagents were purchased

from commercial suppliers (Acros, Novabiochem,

Sigma-Aldrich) and used without purification.

Analytical thin layer chromatography (TLC) was

carried out on Merck precoated silica gel plates

(60F-254) using ultraviolet light irradiation at 254

nm or phosphomolybdic acid solution as staining

reagent (1 wt% in EtOH). Flash column

chromatography (FCC) was performed using silica

gel (J. T. Baker, particle size 40 μm – pore size

60 Å) under a pressure of 0.3-0.5 bar. Analytical

HPLC was performed on an Agilent 1100 system

using a C18 gravity 3 μm reverse phase column

(Macherey & Nagel, Düren, Germany). The

separations were started at 10% MeCN (with 0.1%

HCOOH) in H2O (with 0.1% TFA) with a flow of

1 mL/min, and the MeCN proportion was linearly

increased after 1 min to 100% over a period of

10 min and then kept at constant ratio for a period

of 5 min. Preparative HPLC was performed on a

Varian system using a C18 gravity 5 μm reversed

phase column (Macherey & Nagel, Düren,

Germany) using a MeCN in H2O gradient. 1H- and 13C-NMR spectra were recorded on a Varian

Mercury VX 400 (400.1 MHz (1H) and 100.6 MHz

(13C)) spectrometer. Chemical shifts are expressed

in parts per million (ppm) and the spectra are

calibrated to residual solvent signals of CDCl3

(7.26 ppm (1H) and 77.0 ppm (13C)), CD3OD (3.31

ppm (1H) and 49.0 ppm (13C)) respectively.

Coupling constants are given in Hertz (Hz) and the

following notations indicate the multiplicity of the

signals: s (singlet), d (doublet), t (triplet), q

(quartet), qui (quintet), sext (sextet), sept (septet),

m (multiplet), app (apparent), br (broad signal).

Unless otherwise stated, NMR-spectra were

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recorded at 27°C. Low Resolution Mass Spectra

were recorded on a Thermo Finnigan LCQ ESI

spectrometer (source voltage 70 keV). High

Resolution FAB Spectra were recorded on a Jeol

SX 102 A (matrix: meta-nitrobenzyl alcohol).

High Resolution ESI Spectra were recorded on a

Thermo Electron LTQ Orbitrap (source voltage

3.8 kV, resolution: 60000) spectrometer.

Dihydrotestosterone-derived imidazolide (7)

Dihydrotestosterone (3, 300 mg, 1.00 mmol) and

carbonyl diimidazole (330 mg, 2.00 mmol) was

dissolved in THF (15 mL) and stirred overnight at

room temperature. After disappearance of

dihydrotestosterone on TLC (Rf = 0.4,

EtOAc/hexanes 1:1), the solvent was evaporated.

The residue was partitioned with ethyl acetate

(50 mL) and water (20 mL). The layers were

separated, and the organic extract was washed with

saturated NaHCO3 solution and brine (20 mL

each), dried with MgSO4, and evaporated to obtain

imidazolide 7 a colorless solid (350 mg). The

crude product was purified by FCC

(EtOAc/hexanes 1:9 → 1:2) to obtain the title

compound as a colorless solid (280 mg, yield

73%). TLC: Rf = 0.25 (EtOAc/hexanes 1:1).

HPLC: tR = 10.6 min. ESI-MS: calc. for

C23H33N2O3 385.3, found 385.1 [M+H+].

1H NMR

(400 MHz, CDCl3): δ = 8.13 (1H, t, J = 0.78 Hz,

imidazole), 7.41 (1H, t, J = 1.39 Hz, imidazole),

7.08 (1H, dd, J = 1.4 Hz and 0.79 Hz, imidazole),

4.83 (1H, dd, J = 7.3 Hz and 9.3Hz, H-17 of

dihydrotestosterone), 2.37-2.17 (4H, m), 2.3 (1H,

ddd, J = 15 Hz, 3.98 Hz and 2.18 Hz), 1.95 (1H,

ddd, J = 13.2 Hz, 6.4 Hz and 2.8 Hz), 1.85 (1H, td,

J = 11.9 Hz and 2.9 Hz), 1.70-1.54 (4H, m), 1.53-

1,43 (2H, m), 1.41-1.16 (7H, m), 1.12-1.01 (1H,

m), 0.97 (3H, s, Me), 0.93-0,81 (1H, m), 0.85 (3H,

s, Me). 13C NMR (100 MHz, CDCl3): δ = 211.8

(CO), 148.8 (carbamate), 137.2, 130.8, 117.3 (3C,

imidazole), 87.1 (C-17 of dihydrotestosterone),

53.9, 50.6, 46.8, 44.8, 43.3, 38.7, 38.3, 37.0, 35.9,

35.4, 31.4, 28.9, 27.6, 23.7, 21.1, 12.6, 11.7.

Dihydrotestosterone-derived PEG-carbamate (9)

Imidazolide 7 (50 mg, 0.13 mmol) was added to a

solution of 4,7,10-trioxatridecan-1,13-diamine

(0.085 mL, 0.39 mmol) in acetonitrile (3 mL),

followed by one drop of DMF to get a

homogeneous solution. The reaction mixture was

stirred overnight at room temperature under argon

atmosphere. After complete disappearance of the

starting material on TLC the solvent was

evaporated. The residue was partitioned with ethyl

acetate (20 mL) and water (10 mL). The organic

layer was washed with saturated NaHCO3 solution

and brine (10 mL each), dried with MgSO4 and

evaporated to obtain carbamate 9 a colourless oil

(50 mg). The product was used for the next

reaction without further purification. HPLC: tR =

6.4 min. ESI-MS: calc. for C30H53N2O6 537.4,

found 537.5 [M+H+].

Dihydrotestosterone-derived FITC conjugate (11)

Compound 9 (50 mg crude, 0.13 mmol) was

dissolved in DMF (5 mL) and fluoresceine-5-

isothiocyanate (FITC, 162 mg, 0.39 mmol) and

EtNiPr2 (116 L, 0.65 mmol) was added. The

mixture was stirred and turnover monitored by

HPLC. After 4h, the solvent was evaporated and

the residue was purified using preparative HPLC.

Pure fractions were combined and lyophilized to

give the title compound 11 as an orange solid (14

mg, 12% yield after 2 steps). M.p.: 155-157 °C.

HPLC: tR = 7.9 min. HRMS (ESI): calc. for

C51H64O11N3S 926.4262, found 926.4263. IR (neat,

cm-1): = 3068 (br), 2930, 2871, 1676, 1633. 1H

NMR (400 MHz, DMSO-d6): δ 10.14 (1H, br),

9.96 (1H, br), 9.05 (1H, s), 8.22 (1H, s), 8.11 (1H,

br), 7.73 (1H, br), 7.68 (1H, br), 7.24 (1H, s), 7.16

(1H, d, J = 8.3 Hz), 7.11 (1H, s), 6.98 (1H, s), 6.91

7

9

11

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An olfactory receptor influences melanocyte pigmentation

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(1H, t, J =5.8 Hz), 6.67 (2H, d, J = 2 Hz), 6.60-

6.53 (3H, m), 4.37 (1H, t, J = 8.5 Hz), 3.55-3.45

(12H, m), 2.99 (2H, m), 2.42-2.24 (1H, m), 2.08-

1.84 (4H, m), 1.84-1.77 (2H, m), 1.61-0.96 (17H,

m), 0.95 (3H, s, Me), 0.92-0.79 (1H, m), 0.72 (3H,

s).

Dehydrotestosterone-derived imidazolide (6)

Dehydrotestosterone (2, 30 mg, 0.10 mmol) was

treated similarly to dihydrotestosterone (see 2.).

The imidazolide 6 was obtained as a colorless

solid (33 mg, 0.087 mmol, 87% yield) and was

used directly for the next step. TLC: Rf = 0.27

(EtOAc/hexanes 1:1). HPLC: tR = 9.5 min. ESI-

MS: calc. for C23H29N2O3 381.2, found 381.1

[M+H+].

Dehydrotestosterone-derived PEG-carbamate (8)

Imidazolide 6 (33 mg, 0.087 mmol) was converted

to the PEG-carbamate similar to 7 (see 3.). The

carbamate 8 such obtained was directly used for

the next transformation (yield 48 mg, quant.).

HPLC: tR = 6.5 min. ESI-MS: calc. for C30H49N2O6

533.4, found 533.5 [M+H+].

Dehydrotestosterone-derived FITC conjugate (10)

Compound 8 (0.087 mmol) was dissolved in DMF

(1 mL) and EtNiPr2 (31 µL, 0.17 mmol) was

added. In another flask, 72 mg of FITC (0.17

mmol) was dissolved in DMF (1 mL) and EtNiPr2

(31 µL, 0.17 mmol) was added. The solutions were

combined and more DMF (2 mL) was added. The

orange mixture was stirred at 20°C for 12 h. The

solvent was evaporated and tehresidue subjected to

purification by preparative HPLC. Appropriate

fractions were lyophilized to obtain the title

compound 10 as an orange solid (20 mg, 25 %

over three steps). M.p.: 157-158°C. HPLC: tR = 8.7

min. HRMS (ESI): calc. for C51H60O11N3S

922.3943, found 922.3949 [M+H+]. IR (neat, cm-

1): 3065 (br), 2943, 2871, 1675, 1636. 1H NMR

(400 MHz, DMSO-d6): δ 10.09 (2H, br), 9.89 (1H,

br), 8.19 (1H, s), 8.05 (1H, br), 7.70 (1H, br), 7.15

(1H, d, J =8.4 Hz), 6.96 (1H, t, J =7Hz), 6.6557

(1H, s), 6.6512 (1H, s), 6.60-6.52 (4H, m), 6.12

(2H, m), 5.59 (1H, m), 4.42 (1H, t, J =7.9 Hz),

3.53-3.43 (13H, m), 3.36 (2H, t, J =6.2 Hz), 3.02

(2H, qu, J =6Hz), 2.25-2.14 (2H, m), 2.09-1.98

(1H, m), 1.94-1.86 (1H, m), 1.85-1.72 (3H, m),

1.70-1.56 (4H, m), 1.51-1.26 (4H, m), 1.26-1.07

(3H, m), 1.04 (3H, s), 0.80 (3H, s). 13C NMR (100

MHz, CDCl3): δ 198.7, 169.2, 163.8, 160.2, 156.9,

152.6, 141.0, 129.7, 128.3, 123.7, 113.3, 110.4,

102.9, 81.8, 70.4, 70.3, 70.2, 68.9, 68.7, 50.8, 48.0,

43.6, 38.3, 38.2, 37.6, 36.8, 36.2, 34.3, 34.0, 30.3,

29.3, 28.0, 23.2, 20.5, 16.7, 12.5.

RESULTS

Olfactory receptors are expressed in human

melanocytes- Frog melanophores have previously

been shown to disperse their melanosomes in

response to odorants, although the concurrent

increase in intracellular cAMP levels on pigment

dispersion was discussed controversially (30,31).

Moreover, screening for naturally occurring

compounds with antiproliferative activity on

melanoma cells has led to the identification of the

odorant 4-allyl-2-methoxyphenol (eugenol)

(32,33).

We therefore analyzed whether ORs are

expressed in melanocytes. We started by analyzing

receptor expression in cultured human

melanocytes by reverse-transcriptase PCR (RT-

PCR) (Fig. 1A) and found different ORs to be

expressed, among them OR51E2, an olfactory

receptor which was previously also detected in

prostate cells and in the olfactory epithelium (11).

Since the OR51E2 ligand, the isoprenoid β-ionone

(with an odor of violet), was included in the earlier

studies on the effect of odorants on pigment

dispersion (30,31), we specifically studied the

expression of this receptor in detail. RT-PCR with

8

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intron-spanning primers to exclude amplification

from genomic DNA contamination revealed that

OR51E2 transcripts are expressed in human

epidermal melanocytes (NHEM) from donors of

ethnic origins with differing pigmentation levels

(Fig. 1B). We also detected OR51E2 protein in

human epidermal melanocytes by Western

Blotting and immunocytochemical analysis using

an OR51E2-specific antibody (Fig. 1C,D).

Specificity of the antibody was demonstrated by

co-immunocytochemical staining of Hana3a cells

heterologously expressing rho-tagged OR51E2

(Fig. 1E).

Effect of β-ionone on proliferation and

apoptosis- Because OR51E2 has been shown to be

involved in the regulation of prostate cancer cell

growth (11), we investigated the effect of the

OR51E2 ligand β-ionone on melanocyte

proliferation. Melanocytes were treated for six

days in basal medium containing different

concentrations of β-ionone, and the DNA content

was determined to reveal the number of cells. β-

ionone stimulation decreased the cell number in a

significant manner, even at submicromolar

concentrations (Fig. 2A). The maximal decrease in

proliferation (~30%) was observed after six days

treatment with 50 µM β-ionone. This result was

verified via immunocytochemical staining with an

antibody against the proliferating cell nuclear

antigen (PCNA) as a marker for cell proliferation

(Fig. 2B).

As we observed decreased cell numbers upon

exposure to β-ionone, we investigated the

influence of β-ionone on cell apoptosis rates in

primary human melanocytes. Primary melanocytes

were stimulated for 72 h prior to detection of

apoptosis by assessment of Caspase 3/7 activity as

indicatives of an activated apoptotic signaling

pathway, and TUNEL staining to quantify

apoptotic cells (Fig. 2C). The Caspase 3/7 assay

showed a nominal increase of the caspase activity

after stimulation with 50 µM -ionone, which was

not significant. However, TUNEL staining

implicate that long-term -ionone stimulation does

not induce apoptosis in melanocytes. We therefore

conclude that the observed decrease in cell number

after stimulation with 50 µM -ionone is a result

of a declining proliferation rate.

β-ionone induces melanogenesis and

differentiation- We next investigated the effect of

β-ionone on melanogenesis and dendritogenesis as

indicators of pigment cell differentiation (34). To

observe whether β-ionone stimulation induces

melanogenesis in normal melanocytes, the melanin

content of normal human melanocytes was

determined. Forskolin, a potent stimulator of

adenylate cyclases, which causes melanogenesis

and dendritogenesis of mammalian pigment cells

(35,36), served as positive control. The β-ionone

induced increase of the melanin content was

similar to that of forskolin treatment after 72 h

cultivation in basal medium containing the

respective stimuli (Fig. 2D). Co-stimulation with

the OR51E2 competitive antagonist α-ionone (11)

demonstrates that the observed increase in

melanogenesis depends on activation of OR51E2.

We furthermore identified PKA as a key mediator

of OR51E2-triggered melanogenesis by using the

specific PKA inhibitor H89 (37), which inhibited

the β-ionone-induced melanogenesis when the

cells were pre-stimulated. Concordantly, qPCR

analysis revealed that -ionone stimulation for 72

h induced tyrosinase expression (Fig. 2E), a

promoter of melanocyte melanogenesis. This was

confirmed with Western Blot analysis that showed

an increase in tyrosinase protein levels (Fig. 2F).

Moreover, we examined the effect of β-ionone on

melanocyte morphology as an indicator for cell

differentiation. Melanocyte differentiation

involves branching and extension of the dendrites.

Primary melanocytes cultured in basal medium

were treated for 6 days with 50 µM β-ionone, and

the number of dendrites was quantified. The

majority of control cells exhibited a typical bipolar

morphology, whereas most of the β-ionone treated

cells had multiple dendrites. Quantification of this

effect showed that treatment with β-ionone

induced a morphological change in dendrites and

significantly increased the percentage of cells with

more than two dendrites (Fig. 2G).

As melanocyte cellular responses to external

growth and differentiation signals can be mediated

by MAP Kinases (38-40), we studied activation

(phosphorylation) of prominent members of the

MAP kinase family in β-ionone treated

melanocytes. Western Blot analysis using

antibodies specific for phosphorylated and non-

phosphorylated extracellular stress-regulated

kinase (p44/42 MAPK) and p38 MAPK revealed

that both kinases are activated in β-ionone treated

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melanocytes (Fig. 2H). β-ionone stimulation did

not result in a marked increase in phosphorylation

of SAPK/JNK (data not shown).

In summary, we could show that activation of

OR51E2 affects pigment cell melanogenesis and

differentiation by activation of a cAMP-PKA

mediated pathway.

OR51E2 activation in melanocytes- In order

to investigate if -ionone exerts its effects on

melanocytes by activation of OR51E2 we first

examined the effects of short-term β-ionone

stimulation on the level of intracellular Ca2+ in

melanocytes via the Ca2+ imaging method.

Activation of an OR expressed in olfactory

sensory neurons, as well as in heterologous cell

systems, induces intracellular Ca2+ signals as a

result of OR-initiated signaling. Stimulation of

Fura-2 loaded melanocytes with β-ionone also

caused a rise in cytosolic Ca2+ (Fig. 3A). However,

this β-ionone induced Ca2+ signal was different in

response kinetic compared to olfactory sensory

neurons and Hana3a cells expressing OR51E2,

respectively. Olfactory sensory neurons and OR

expressing Hana3a cells show a fast transient Ca2+

signal upon odorant-ligand stimulation, whereas

application of β-ionone in melanocytes caused a

slow but robust increase in cytosolic Ca2+ that

started rising after 1 min from the begin of the

application (Fig. 3B). When the ligand was

supplied continuously, the maximal amplitude was

reached after 5 min application of β-ionone. The

signal decreased afterwards in the presence of the

ligand, indicating a desensitization of the signaling

cascade (Fig. 3C). The β-ionone induced rise in

intracellular Ca2+ was found to be dose-dependent,

and the threshold concentration to trigger a cellular

response to β-ionone was approximately 10 µM

(Fig. 3D).

We next aimed to confirm that the β-ionone

induced increase in cytosolic Ca2+ is dependent on

activation of OR51E2. We therefore performed

Ca2+ imaging experiments with reduced OR51E2

expression rate due to RNA silencing (Fig. 3E). β-

ionone induced Ca2+ signal amplitudes were

quantified and compared to those in non-

transfected control cells within the same

experiment. The average of the β-ionone mediated

Ca2+ signal amplitude was found to be

significantly reduced (~85%) in siRNA-expressing

melanocytes compared to control cells (Fig. 3F,G).

Expression of scrambled control siRNA did not

show an effect on the β-ionone induced Ca2+

responses in transfected cells; neither did the

transfection procedure alter Ca2+ responses of

transfected melanocytes cells to Endothelin-1

(Fig. 3G), which was reported to induce a Ca2+ rise

in melanocytes through action on the Endothelin-

1B receptor (41). We further showed that the β-

ionone evoked Ca2+ rise in melanocytes could be

prevented by co-application of the OR51E2

competitive antagonist α-ionone (11) (Fig. 3I);

whereas α-ionone alone did not produce Ca2+

signals in melanocytes (Fig. 3H). Together, these

data show that β-ionone produces an increase in

the cytosolic Ca2+ concentration in melanocytes,

and that the β-ionone induced Ca2+ rise is mediated

by the activation of OR51E2.

OR51E2 signaling in melanocytes- We next aimed

to elucidate the signaling mechanism of OR51E2

in primary human melanocytes. To determine the

origin of the agonist-evoked Ca2+ rise, we used

Ringer’s solution with varying Ca2+

concentrations. The β-ionone-induced Ca2+

increase still occurred after removal of the

extracellular Ca2+, suggesting that release from

intracellular stores contributes to the β-ionone

induced Ca2+ signal (Fig. 4A,G). The signal

amplitude was significantly reduced (by ~30%),

the signal onset was delayed, and the rise in

intracellular Ca2+ was slower under Ca2+-free

conditions. Extracellular Ca2+ is therefore

considered to significantly account for the β-

ionone induced Ca2+ response in melanocytes. The

observation that Thapsigargin prestimulation

suppresses the β-ionone induced Ca2+ response

(Fig. 4B,G) might reflect a reciprocal regulation

between the OR51E2 initiated pathway and store-

operated calcium entry (SOCE). In order to

identify potential Ca2+ channels mediating the Ca2+

influx, we tested pharmacological inhibitors of

SOCE and TRP channels. Whereas ORAI and

TRP channel blocker 2-APB significantly

diminished the β-ionone induced Ca2+ signal (Fig.

4C,G), TRPC and SOCE channel blocker SKF

96365 (Fig. 4D,G), SOCE inhibitor BTP2 (Fig.

4E,G) as well as stretch-activated calcium channel,

TRPA1 and TRPC blocker GdCl3 (Fig. 4E,G) did

not influence the β-ionone induced Ca2+ rise in

melanocytes when co-applied with β-ionone.

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In olfactory sensory neurons, ORs couple to Gαolf,

a protein homologous to Gαs, resulting in

activation of an adenylate cyclase and generation

of cAMP. We here investigated, whether OR51E2

activation in melanocytes also modulates cellular

cAMP levels. We determined cAMP levels in

primary melanocytes and found that β-ionone

stimulation increased the intracellular cAMP

concentration fourfold. This effect was suppressed

by co-stimulation with the OR51E2 inhibitor α-

ionone, indicating that the elevation of cAMP

levels upon β-ionone treatment is caused by

activation of OR51E2 (Fig. 4H). Usage of

pharmacological tools that specifically inhibit key

enzymes typically activated by GPCR stimulation

(adenylate cyclase, phospholipase C) did not allow

for straightforward interpretation of the Ca2+ data,

probably due to the observed cytotoxic side effects

of the tested inhibitors MDL, edelfosin and

U73122 in melanocytes. However, the

pharmacological characterization using inhibitors

of Ca2+ signaling (Thapsigargin, 2-APB, SKF,

BTP2, GdCl3,) indicate that the OR51E2-triggered

Ca2+ signal is constituted by Ca2+ release from

intracellular stores and an additive Ca2+ influx

from the extracellular space. Ca2+ influx may be

mediated by members of the transient receptor

potential (TRP) ion channel family that are

expressed in melanocytes such as TRPM family

members (42), TRPA1 (43) or TRPV1 (44) or

calcium release-activated channels, such as

ORAI1/2 (45). However, an involvement of ORAI

channels, TRPC channels and TRPA1 could be

excluded because SKF, BTP2 and GdCl3 did not

block the β-ionone-induced Ca2+ signal at tested

concentrations. Therefore, we suggest that

members of the TRPM family are mediating the

observed Ca2+ influx. This assumption is supported

by the finding that 2-APB, which blocks inter alia

TRPM3, TRPM7 and TRPM8, significantly

reduced the β-ionone-induced Ca2+ signal.

Subcellular localization of OR51E2- A well

characterized GPCR that is involved in biogenesis

of the pigment producing organelles, the

melanosomes, is the ocular albinism type 1 (OA1)

(46-51). OA1, an atypical GPCR, primarily

localizes to intracellular endolysosomes and the

melanosomes of retinal pigment epithelial cells

rather than the cell surface (52,53).

We therefore investigated where OR51E2

resides in human epidermal melanocytes. In order

to determine the subcellular localization of the

receptor, we performed immunofluorescence

studies to visualize OR51E2 protein. Melanocytes

were labeled with an antibody against OR51E2. In

epidermal melanocytes, immunofluorescence

staining revealed that OR51E2 is localized in a

vesicular pattern throughout the cytosol (Fig. 5A).

OR51E2 containing vesicles accumulated at the

extremity of the dendrites. Localization at the

plasma membrane was not apparent in

immunocytochemical stainings. Co-staining with

antibodies targeting several cellular organelles

revealed that OR51E2 did not localize to the

endoplasmatic reticulum, the Golgi apparatus,

melanosomes and lysosomes (data not shown), but

showed co-localization with EEA-1 (Fig. 5B), an

early endosomal marker. Analysis of numerous

cells revealed that there was clear OR51E2

labeling at the plasma membrane.

Since immunocytochemical methods failed to

give clear information about membrane

localization of OR51E2, we next aimed to resolve

whether receptor activation occurs also at the

plasma membrane or only at intracellular

membranes. To provide evidence for plasma

membrane localization of OR51E2 we detected the

protein by western Blot in surface preparations of

primary melanocytes (Fig. 5C).

In order to distinguish whether the -ionone

induced Ca2+ signal results from activation of

surface or cytosolic OR51E2, we generated

ligands for receptor activation, which are less

membrane permeable than the unmodified ligands.

β-ionone exhibits hydrophobic structural

properties and may therefore be membrane

permeable, making it possible that the receptor is

activated by the ligand inside an intracellular

compartment. Other OR51E2 activating ligands

described previously are the steroids 4,6-

androstadien-17α-ol-3-one, 1,4,6-androstadien-

3,17-dione, and 6-dehydrotestosterone (11). For

chemical structures, see Scheme 1. Activation of

OR51E2 by these steroid ligands (100 µM) could

be shown in melanocytes (Fig. 5D), whereas

stimulation with dihydrotestosterone (DHT),

which does not activate recombinant OR51E2,

failed to induce Ca2+ signals in melanocytes.

Notably, the Ca2+ signal induced by OR51E2

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activating steroid ligands was significantly higher

compared to the β-ionone induced Ca2+ responses

(Fig. 5D).

These data suggested that -ionone might be a

weaker agonist, and that the A/B-ring region of the

steroids was important for ligand activity, whereas

the D ring should be amenable to modification. To

study the cellular site of activation of OR51E2, we

synthesized an OR51E2 activating dehydrotestos-

terone appended with the membrane-impermeable

anionic dye molecule fluorescein isothiocyanate

(FITC) by means of a water-soluble PEG linker.

As control, we used DHT that was shown to be

inactive on OR51E2 (11). The FITC label was

attached to the OH-group of dihydrotestosterone

and 6-dehydrotestosterone on position 17 (see

scheme 1 and experimental section). Due to the

change in physico-chemical properties these dye

labeled, hydrophilic steroid ligands are expected to

exhibit a reduced cell membrane permeability

compared to the parental steroids.

Interestingly, the FITC-tagged

dehydrotestosterone derivative was found to

activate recombinant OR51E2 as shown by Ca2+

imaging experiments on Hana3a cells transiently

expressing OR51E2 (Fig. 5E). Similarly,

application of the same FITC-tagged steroid ligand

to primary human melanocytes triggered Ca2+

responses (Fig. 5F,G). Although the maximal

signal amplitude was smaller compared to

untagged 6-dehydrotestosterone, it resembled the

Ca2+ response amplitude evoked by β-ionone.

Neither treatment of melanocytes with the inactive

steroid DHT, nor with FITC-DHT induced a

comparable Ca2+ response in the cells (Fig. 5F,G),

indicating that OR51E2 protein, which is present

at the plasma membrane of melanocytes, received

the external signal.

Repetitive β-ionone stimulation of

melanocytes resulted in an increase in the Ca2+

response amplitudes (Fig. 5H). When we exposed

melanocytes to β-ionone before re-stimulation in

Ca2+ imaging experiments, we observed significant

increases in the β-ionone induced Ca2+ levels that

were dependent on the duration of preexposure

(Fig. 5H,I). We assume that these effects could be

caused by increased insertion of OR51E2 into the

plasma membrane upon activation of the receptor,

thereby increasing the low amounts of functional

OR51E2 at the cell surface of melanocytes.

DISCUSSION

Functionality of an ectopically expressed

odorant receptor in melanocytes- The first

evidence for an influence of odorants on pigment

cells was obtained in the 1980s, when the

superfamily of odorant receptors was unknown.

Frog melanophores were shown to disperse their

melanosomes in response to odorants, and odorant

induced pigment dispersion was accompanied by

rises in intracellular cAMP levels (30).

Corresponding studies discovered cinnamaldehyde

and β-ionone to have pigment dispersing activity

in fish melanophores (31), but both substances did

not result in a measurable rise in cAMP levels. We

describe here that β-ionone lead to an increase in

the Ca2+ concentration and elevated cAMP levels.

In addition, melanin content increased in primary

epidermal melanocytes, possibly due to

upregulated expression of tyrosinase. Moreover,

we describe that OR51E2 mediates β-ionone

induced effects in melanocytes. By gene-silencing

and by using a specific antagonist, we could

clearly show that the β-ionone induced Ca2+ signal

in melanocytes depends on the expression of

OR51E2.

OR51E2 belongs to the family of odorant

receptors and is expressed in prostate cancer cells

(11). Analysis of the role of OR51E2 in

melanocytes revealed that the receptor affects

melanocyte proliferation, differentiation and

melanogenesis, and indicates that ectopically

expressed ORs have important cellular functions.

As OR51E2 expression is upregulated in human

prostate cancer cells compared to healthy prostate

epithelial cells, the receptor may be used as a

potential tumor biomarker (54-57). Therefore, it

would be interesting to investigate OR51E2

expression and function in melanoma cells derived

from epidermal melanocytes. OR51E2 is acting as

a cell surface steroid receptor that mediates rapid,

non-genomic, steroidal signalling in prostate

cancer cells (11). The previously identified steroid

ligands of OR51E2 activate Ca2+ responses in

melanocytes in a similar fashion. The exquisite

selectivity of this receptor with respect to the

molecular structure of the agonist suggests that

endogenous ligands should exist to regulate the

proliferation and differentiation of melanocytes. It

is tempting to speculate that this yet elusive

endogenous ligand for OR51E2 may be a

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keratinocyte-derived factor that is chemically

similar to the identified agonists, possibly a

steroidal compound.

OR51E2 localization- Intracellular

localization of GPCRs was previously shown in

retinal pigment epithelial cells. The atypical GPCR

OA1 is localized to membranes of melanosomes

and late endosomes/lysosomes, although low

amounts of the receptor were detected also at the

plasma membrane (58). OA1 is considered to

regulate melanosome biogenesis by transducing

signals through activation of heterotrimeric G-

proteins on the cytoplasmatic side of the organelle

membrane (46,48,59).

OR51E2 was mainly localized in early

endosomes associated with EEA-1 in human

epidermal melanocytes. Endosomal organelles are

direct precursors to premelanosomes (stage I

melanosomes) (60). As in other cells, two distinct

early endosomal domains can be distinguished,

tubulovesicular structures containing the bulk of

EEA-1, and globular structures, which hold

enriched concentrations of the melanoma-

associated protein melan-A. However, we could

exclude localization of OR51E2 to the globular

endosomal domains by co-labeling OR51E2 and

melan-A. Although immunocytochemical signals

were not apparent in immunocytochemical

analysis, we clearly demonstrated plasma

membrane localization of OR51E2 by cell surface

preparations and western blot analysis. Activation

of OR51E2 in melanocytes still occurred when

using a steroid agonist for OR51E2 with reduced

membrane permeability, indicating that the

observed Ca2+ signals results from activation of

OR51E2 protein at the cell surface rather than

cytosolic OR51E2.

Signaling cascade of OR51E2 in normal

human epidermal melanocytes- The present study

shows that signaling of OR51E2 in melanocytes

involves cAMP, potentially TRPM family

members, Ca2+ and activation of PKA and

MAPKs. The OR51E2 induced Ca2+ signal was

found to be partially dependent on extracellular

Ca2+, as the amplitude of the response was

significantly reduced in the absence of Ca2+.

However, pharmacological characterization

revealed that the influx of extracellular Ca2+ is

neither mediated via store-operated Ca2+ channels

of the ORAI family, nor by TRPC, TRPV and

TRPA1 channels, but indicate an involvement of

other TRP channels. Based on previous reports and

our results (with given limitations of the employed

Ca2+ imaging technique), we conclude that the

molecular identity of the TRP channels is in the

TRPM family as members of this TRP family are

expressed in melanocytes and can be blocked by 2-

APB while remaining unaffected by the other

tested inhibitors.

The proposed OR51E2 initiated signal

transduction pathway in melanocytes, may involve

PLC-mediated signaling as well. Unfortunately,

common inhibitors of PLC appeared unsuitable for

application on melanocytes. In classical olfactory

tissues, olfactory receptors can couple to both,

adenylyl cyclase and PLC mediated pathways

(61).

In prostate cancer cells, OR51E2 activates

TRPV6 via Src-kinase in a G-protein independent

manner (62). However, comparison of the

OR51E2 signaling in prostate cancer cells and

melanocytes reveals differences, such as an

involvement of adenylate cyclase (11). Thus, the

signal transduction cascade of ectopically

expressed ORs seems to be majorly determined by

the cellular background, and not by the properties

of the receptor protein.

Role of OR51E2 on melanogenesis and

pigment cell differentiation- Pigment production in

vertebrates is modulated by a variety of hormones

and neurotransmitters acting on transmembrane

receptors located on the cell surface (63,64).

Activation of the ET-1 receptor increases cytosolic

Ca2+ concentrations via ORAI1 channels, thereby

stimulating melanogenesis (65,66). The

intracellular Ca2+ concentration itself was found to

be not essential for melanogenesis, but to play an

important role in modulating the responses of

melanocytes to melanogenic stimuli (67,68).

Activation of OR51E2 resulted in a prolonged

increase in the intracellular Ca2+ concentration,

and to an induction of melanogenesis via the

cAMP pathway. Regulation of melanogenesis was

shown to involve stimulation of adenylate cyclase,

followed by an increase in the intracellular cAMP

level and activation of cAMP-dependent protein

kinases (PKA) (69). Activated PKA is involved in

the phosphorylation of cAMP responsive element

binding protein (CREB) and CREB binding

protein. Phosphorylated CREB leads to an

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activation of microphthalmia-associated

transcription factor (MITF), whereas MITF

regulates the expression (but not activity) of

melanogenic enzymes (tyrosinase, TYRP1 and

TYRP2) (reviewed in 70). Thus, the observed

upregulation of tyrosinase likely results from

OR51E2 triggered activation of PKA.

Downstream signaling of OR51E2 in

epidermal melanocytes also involves activation of

p38 MAPK and p44/42 MAPK (ERK1/2), as

possible modulators of melanogenesis. These

MAPKs are described to control pigment cellular

responses to melanogenetic stimuli by induction of

tyrosinase proteosomal degradation, thus

antagonizing activated cAMP signalling (71).

Effects of OR51E2 activation on melanocyte

morphology and melanogenesis implicate that

OR51E2 is also involved in regulation of

melanocytes differentiation. Many coat color

genes, known for their ability to regulate

melanosome formation, control in addition

melanoblast migration, proliferation and

differentiation, and melanosome distribution.

Thus, melanocyte proliferation and differentiation

is not only regulated by typical growth factor

receptors, but also by genes classically known for

their role in pigment formation.

Interestingly, repetitive cell stimulation in

Ca2+ imaging experiments showed an increased

responsiveness of melanocytes pretreated with β-

ionone. We hypothesize that OR51E2 that is

localized in the early endosome may translocate to

the plasma membrane upon activation of cell

surface receptors. A new paradigm is emerging in

some cellular contexts, in which stocks of

functional GPCRs retained within intracellular

compartments can be rapidly mobilized to the

plasma membrane to maintain sustained

physiological responsiveness (72). However,

further data must be acquired to confirm such a

role for OR51E2 in human melanocytes.

The present study constitutes a new example

for the functionality of ectopic ORs. It also

contributes to the understanding the molecular

processes involved in regulation of skin

pigmentation by showing that the ectopically

expressed olfactory receptor OR51E2 is

functionally expressed in melanocytes. Pigment

cells respond to the OR51E2 ligand with decreased

proliferation rates and increased melanogenesis.

As OR51E2 induced signaling mechanisms could

influence melanocyte homeostasis, receptor

activating steroids or terpenoids might provide

novel compounds for the treatment of

pigmentation disorders and proliferative pigment

cell disorders such as melanoma.

Acknowledgments- We thank F. Moessler and H. Bartel for their technical assistance, Dr. J. Panten

(Symrise, Holzminden, D) for providing odorants, Dr. G. Neufang and W. Gerwat (Beiersdorf, Hamburg,

D) for providing primary melanocytes, Dr. W. Zhang (Bayer HealthCare, CHN) for her contributions to

the preliminary experiments in this study, and Dr. S. Sinclair (Ruhr-University Bochum, D) for proof

reading and helpful suggestions on the manuscript.

Conflict of interest- The authors declare that they have no conflicts of interest with the contents of this

article

Author contributions- L.G., N.J. and S.V. performed experimental research. H.-D.A. performed synthesis

of the FITC-steroids. L.G., E.M.N. and H.H. conceived and designed the research. L.G., N.J. and E.M.N.

wrote the manuscript.

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FOOTNOTES

*This study was supported by grants from the Deutsche Forschungsgemeinschaft to E.M.N. and H.H.

(SFB642). N.J. is funded by the Heinrich-and-Alma-Vogelsang-Stiftung. ‡Cell Physiology, Ruhr-University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany §Organic Chemistry I, Friedrich Schiller University, Humboldtstr. 10, 07743 Jena, Germany †Pharmacology and Toxicology, University Hospital Jena, Drackendorfer Str. 1, 07747 Jena, Germany ¥New Address: Department of Chemistry, IIT Guwahati, North Guwahati, Assam - 781 039, India 1Both authors contributed equally 2To whom correspondence should be addressed: Cell Physiology, Ruhr-University Bochum,

Universitaetsstrasse 150, 44801 Bochum, Germany, lian.gelis@rub.de, tel +49-234-3224586

The abbreviations used are: ΔCt, cycle threshold; 2-APB, aminoethoxydiphenyl borate; cAMP, cyclic

adenosine monophosphate; CREB, cAMP responsive element binding protein; DHT, dehydrotestosterone;

DMSO, dimethyl sulfoxide; EEA-1, early endosome antigen 1; EGTA, ethylene glycol tetraacetic acid;

FITC, fluorescein isothiocyanate; GAPDH, glycerinaldehyde-3-phosphate-dehydrogenase; GPCR, G-

protein coupled receptor; MAPK, mitogen-activated protein kinase; MITF, microphthalmia-associated

transcription factor; NHEM, neonatal normal human epidermal melanocytes; OR, olfactory receptor;

ORAI, calcium release-activated calcium channel; PCNA, proliferating cell nuclear antigen; PEG,

polyethylene glycol; PSGR, prostate-specific G-protein coupled receptor; SERCA,

sarcoplasmic/endoplasmic reticulum calcium ATPase; TRP, transient receptor potential ion channel;

SOCE, store-operated calcium entry; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end

labeling

FIGURE LEGENDS

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FIGURE 1. OR51E2 is expressed in human epidermal melanocytes. (A) RT-PCR detection of 13

different ORs in normal human epidermal melanocytes (NHEM). Gel electrophoresis of OR amplicons

from melanocyte cDNA (+) and no reverse transcriptase cDNA controls (-) to exclude genomic DNA

contamination. Amplification of Actin and tyrosinase-related protein 1 (Tyr-P1) served as positive

controls. OR51B5, OR5P3 and OR6B3 transcripts were not detected. (B) Detection of OR51E2 transcripts

in melanocytes (NHEM) by RT-PCR, together with a schematic representation of the intron overspanning

primers for this receptor. OR51E2 transcripts could be detected in melanocytes of different pigmentation

levels (caucasian and negroid origin). LNCaP cells served as positive control, negative control: genomic

DNA (genDNA). The size of the OR51E2 amplicon was 1050 bp (cDNA) or >15 000 bp (genDNA). (C)

Detection of OR51E2 protein in primary melanocytes by Western Blotting, the size of the OR51E2

monomeric (51E2-M) protein is 35 kDa and dimeric 70 kDa (51E2-D). (D) Immunocytochemical staining

using an OR51E2 specific antibody, shown are confocal micrographs of an OR51E2 staining in

melanocytes. The control consisted of omission of the primary antibody. Bars indicate 15 µM. (E) Co-

immunocytochemical staining of Hana3a cells transiently expressing OR51E2 with an N-terminal rho-tag,

which consists of the first 20 amino acids of rhodopsin. Detection of the recombinant rho-OR51E2-

protein was performed using an antibody against OR51E2 and an antibody against the n-terminal rho-tag.

Shown are confocal micrographs of the OR51E2 staining in OR51E2 expressing Hana3a cells. Mock-

transfected Hana3a cells served as negative control. Bars indicate 20 µM. Cell nuclei were stained with

4’,6-diamidino-2-phenylindole (DAPI). Co-labeling (yellow) of OR51E2-expressing Hana3a cells

indicates specificity of the used OR51E2-antibody.

FIGURE 2. Activation of OR51E2 promotes differentiation. (A) β-ionone stimulation affects

melanocytes proliferation. Proliferation of melanocytes after treatment with increasing concentrations of

β-ionone for 6 days compared to control conditions. The relative cell number was determined by

measurement of the DNA content. The data are shown as the mean of three independent experiments with

five technical replicates and were normalized to the cell number in control experiments, significance was

calculated by Student’s t-test referring to cell number of untreated control cells. (B) Immunocytochemical

staining using an antibody against PCNA reveals significantly reduced numbers of proliferating cells after

stimulation with β-ionone compared to control cells. Left panel: Immunofluorescence confocal

micrographs of melanocytes labeled with a PCNA-specific antibody (green) and Alexa Fluor® 546

Phalloidin (red). Melanocytes were treated with 50 µM β-ionone or solvent only (control) for 6 days. Bar

indicates 50 µm. Right panel: the data are shown as the mean of four independent experiments each with

500 quantified cells, which were normalized to the cell number in control experiments. Significance was

calculated by Student’s t-test referring to cell number of untreated control cells. (C) β-ionone stimulation

has no significant effect on melanocyte apoptosis. Left panel: Induction of apoptotic pathways was

assessed by the Capase-Glo 3/7 assay. Significance was calculated by Student’s t-test referring to cells

treated with the solvent only. Right panel: Representative photomicrographs of melanocytes stained by

TUNEL assay after treatment with 50 µM β-ionone for 72 h. DNAseI treatment (20 min) served as

positive control for the detection of apoptosis. (D) β-ionone stimulation induces melanogenesis. Melanin

content was measured 72 h after β-ionone treatment (50 µM). The β-ionone-induced effect on

pigmentation was supressed by co-stimulation with α-ionone (200µM) or prestimulation with H89 (10

µM) for 2h.The data are the average of results from three biological replicates each performed in

triplicate, and were normalized to the melanin content in solvent only treated cells (0.1 % DMSO,

control). Forskolin (20 µM) served as a positive control. Error bars indicate the SEM. Significance was

calculated by Student’s t-test referring to the melanin content in control cells. (E) RT-qPCR quantification

of tyrosinase gene expression in melanocytes stimulated for 6 h with β-ionone (50 µM), forskolin (20 µM)

and solvent only (0.1% DMSO, control). Right panel: Gel electrophoresis of tyrosinase amplicons using

two different primer pairs (1, 2) from melanocyte cDNA (+) and no reverse transcriptase controls (-

).Amplification of GAPDH served as internal control. Left panel: Relative tyrosinase gene expression of

β-ionone stimulated melanocytes using two different primer pairs (1, 2). Tyrosinase mRNA levels were

normalized by GAPDH in each experiment and changes in mRNA expression were calculated compared

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to control cells. Bars represent the mean of three experiments, each performed in triplicate. Error bars

represent the SEM. (F) Right panel: Western Blot analysis reveals -ionone induced increase in

tyrosinase protein. Cells were stimulated with -ionone (50 µM) and forskolin (20 µM, positive control)

for 72 h. Detection of GAPDH served as internal standard. Left panel: Quantification of tyrosinase signal

intensities. Bars represent the mean of four independent experiments, tyrosinase signal intensities were

normalized by GAPDH in the same experiment. (G) Left panel: Phase-contrast image of an

undifferentiated and differentiated melanocyte in culture. Undifferentiated melanocytes are bipolar, less

pigmented and have small cell bodies, whereas differentiated melanocytes exhibit an increased size of the

cell body, increased pigmentation and multiple dendrites. Bar indicates ten µm. Right panel: Exposure to

β-ionone induces melanocyte dendritogenesis. Cells were cultured for 6 d in basal medium containing β-

ionone (50 µM) and 0.1 % DMSO as negative control. Forskolin (20 µM) served as a positive control.

Undifferentiated (grey part of the bar) and differentiated cells (black part of the bar) were counted and are

shown as percentage of total cells. The data are the mean of three independent experiments each with 500

cells quantified. Statistics were performed by Student’s t-test, referring to cell number of control cells. (H)

Activation of OR51E2 results in phosphorylation of p38 MAPK and p44/42 MAPK (ERK1/2) as shown

by Western Blot analyses. Melanocytes were stimulated with 50 µM β-ionone for 30 min. Detection of

p38 MAPK and ERK total protein is shown to control loaded protein amounts. Error bars represent SEM.

FIGURE 3. OR51E2 activation induces a Ca2+ signal. (A) Representative picture of Fura-2 loaded

melanocytes. Intracellular Ca2+ levels are indicated in pseudocolors, images of changes in cytosolic Ca2+

were captured during a representative experiment. (B) Representative Ca2+ imaging trace of a melanocyte.

In a randomly selected field of view, β-ionone (50 µM) application induces a transient, slow rise in

intracellular Ca2+ (black) in individual cells. The grey Ca2+ imaging trace is representative for the vehicle

controls (0.1% DMSO). Application of the solvent did not result in any changes in cytosolic Ca2+.

Cytosolic Ca2+ levels were monitored as integrated f340/f380 fluorescence ratio expressed as a function of

time. (C) In prolonged stimulation the maximal signal amplitude is reached after 5 min application of β-

ionone. After reaching the maximum peak, the cytosolic Ca2+ concentration decreases until a plateau

phase is sustained. β-ionone was applied for 9 min. (D) β-ionone induced Ca2+ increase is dose-dependent.

β-ionone was applied for 9 min at different concentrations to ensure maximal signal amplitude generation.

Signal amplitude was quantified, normalized to the maximal peak height and is displayed as a function of

the applied β-ionone concentration (n=49-129 cells). (E) Ca2+ imaging on primary melanocytes

transfected with a plasmid encoding for siRNA directed against OR51E2. As the plasmid encode for GFP

under the same promoter, siRNA-expressing cells can be identified via GFP fluorescence (left panel),

pseudocolors images captured of Fura-2 loaded cells during Ca2+ imaging experiment (right panel).

OR51E2-siRNA/GFP expressing melanocytes (turquoise labeling) show no significant changes in the

Fura-2 ratio upon application of β-ionone, whereas non-transfected cells (blue labeling) do. (F) β-ionone

induced Ca2+ signals in siRNA transfected (turquoise labeling) and control melanocytes (blue labeling)

displayed as a function of time. 50 µM β-ionone was applied for 5 min. (G) Quantification of the relative

signal amplitudes of the β-ionone evoked Ca2+ signals, normalized to the β-ionone response of control

cells (n= 109 cells), n=4 independent transfections were analyzed. Shown are OR51E2-siRNA expressing

melanocytes (n= 42 siRNA-expressing cells), and melanocytes transfected with a plasmid encoding for

scrambled OR51E2-siRNA (control siRNA, n=27). OR51E2-siRNA transfected (n=23) and control cells

(n=64) were stimulated with 40 nM Endothelin-1 (ET-1) to control that siRNA expression does not affect

cell viability. Error bars represent the SEM. Significance was calculated by Student's t-test for each

sample group referring to the signal amplitude in control melanocytes. (H) Representative Ca2+ imaging

traces of Fura-2 loaded melanocytes. -ionone (250 µM) was applied for 5 min. Cytosolic Ca2+ levels are

monitored as integrated f340/f380 fluorescence ratio expressed as a function of time. (I) Co-application of

the OR51E2-specific inhibitor α-ionone reduces the β-ionone induced Ca2+ signals (n= 48). β-ionone (50

µM) and the α-ionone and β-ionone 2:1 mix (100 µM and 50 µM, respectively) were applied for 4 min.

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FIGURE 4. OR51E2 signaling in melanocytes. (A-F) Representative recordings of Fura-2 loaded

melanocytes. Cytosolic Ca2+ concentration is monitored as integrated fluorescence ratio as a function of

time. (A) Ringer’s solution containing 50 µM EGTA decreases the β-ionone (50 µM) induced response.

(B) Pretreatment with Thapsigargin (10 µM) or (C) 2-APB (25 µM) reduces the β-ionone (50 µM)

induced Ca2+ signals in melanocytes. (D) SKF 96365 (SKF; 10µM) (E) BTP2 (20µM) and (F) GdCl3

(100µM) do not influence the β-ionone (50 µM) induced Ca2+ response in melanocytes. (G) Quantification

of β-ionone-induced Ca2+ signals in blocker experiments relative to control measurements (β-ionone only)

or quantification of Ca2+ signal amplitudes in Ca2+ free conditions (+ 50 µM EGTA), normalized to the

melanocyte responses measured in Ca2+ containing buffer (control). Data significance was calculated

using Student’s t-test referring to the β-ionone induced Ca2+ signal in controls. The data are shown as the

means ± SEM (n≥65 cells). (H) OR51E2 signaling involves activation of adenylate cyclases. β-ionone

(50 µM, 30 min) stimulation resulted in a fourfold increase in intracellular cAMP, that was suppressed by

co-stimulation with α-ionone (200 µM). Stimulation of melanocytes by α-ionone alone did not increase

intracellular cAMP. Forskolin (20 µM) served as a positive control. Experiments were performed as

duplicates in four independent experiments. Significance was calculated by Student’s t-test referring to the

intracellular cAMP concentration in control cells. Error bars represent the SEM.

FIGURE 5. Intracellular localization of OR51E2. (A) Immunofluorescence confocal micrographs of a

melanocyte labeled with an OR51E2-specific antibody (green) and DAPI to visualize the nucleus (blue).

An intracellular, vesicular staining of OR51E2 can be observed. Bar indicates 5 µm. (B)

Immunofluorescence confocal micrographs of a melanocyte co-labeled with an OR51E2-specific antibody

(green) and an antibody to EAA-1 (red). Co-staining of cellular organelles appears in yellow in the

merged image (arrows). Bar indicates 5 µm. (C) Surface localization of OR51E2 in melanocytes verified

by surface biotinylation and detection by Western Blot. A representative western blot of biotinylated

membrane are shown (n=3 independent experiments). GAPDH served to control of purification of cell

surface proteins only. (D) Ca2+ responses of melanocytes to steroid-ligands of OR51E2. Shown are

representative Ca2+ imaging traces of melanocytes stimulated with the OR51E2 steroid ligands 6-

dehydrotestosterone (s1, 2), 4,6-androstadien-17-ol-3-one (s2, 4) and 1,4,6-androstadien-3,17-dione (s3,

5). Ca2+ signals are displayed as integrated fluorescence ratio as a function of time. Steroids were applied

for 5 min. (E) Heterolougsly expressed OR51E2 responds to FITC-tagged 6-dehydrotestosterone. Shown

is a representative Ca2+ imaging trace of a Hana3a cell transiently expressing OR51E2. ATP (20 µM, 2 s)

served to control the ability of the cells to produce Gq mediated Ca2+ signals. The integrated fluorescence

ratio (f340/f380) of the Fura-2 loaded cell is shown as a function of time. FITC-6-dehydrotestosterone

(100 µM) was applied for 10 s. (F) Representative Ca2+ imaging recording of melanocytes treated with

FITC-steroids (100 µM) for 5 min. Only application FITC-6-dehydrotestosterone (FITC-s1) induced a

Ca2+ signal in the cells, application of FITC-DHT shows no effect on cytosolic Ca2+ levels. (G)

Quantification of cytosolic Ca2+ signal amplitudes upon stimulation of melanocytes with β-ionone (50

µM), 6-dehydrotestosterone (s1, 100 µM), dihydrotestosterone (DHT, 100 µM), FITC-6-

dehydrotestosterone (FITC-s1, 100µM), and FITC-DHT (100 µM). Signal amplitudes were quantified and

normalized by the β-ionone induced maximal signal amplitudes. Three independent experiments (each

with > 70 cells) were performed in quadruplicates for each steroid, data are shown as the mean.

Significance was calculated by Student’s t-test for each sample group referring to the β-ionone induced

signal amplitude. Error bars represent the SEM. (H) Repetitive stimulation with β-ionone evokes

increasing Ca2+ signals. Representative Ca2+ imaging trace of a Fura-2 loaded melanocyte. β-ionone (50

µM) was applied for 5 min in each application. (I) Left panel: Different durations of pretreatment with 50

µM β-ionone results in increased responses to consecutive stimulation with β-ionone. Shown are

representative Ca2+ imaging traces of Fura-2 loaded primary melanocytes, β-ionone (50 µM) was

consecutively applied for 5 min. Right panel: Quantification of signal amplitudes in melanocytes

pretreated with β-ionone (50 µM) for different durations, bars represent the mean normalized to control

amplitudes (from cells not pretreated). Three independent experiments were performed for each

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prestimulation, each with > 50 cells. Significance was calculated by Student’s t-test for each sample group

referring to the β-ionone induced Ca2+ signal in control cells. Error bars represent the SEM.

SCHEME 1. Chemical Structures of ligands and synthesis of FITC-labeled steroids. (A) Molecular

structures of ligands used in this study. Steroids are labelled according to IUPAC. The dienone

substructure common to active ligands is highlighted. (B) Synthesis of FITC labelled steroids. i) Alcohol

2 or 3, THF (70 mM), carbonyldiimidazole (2.0 equiv.), 12 h, 20°C; ii) monoimidazolide 6 or 7,

acetonitrile/DMF (99:1), 4,7,10-trioxatridecan-1,13-diamine (3.0 equiv.), 12 h, 20°C; iii) amine 8 or 9,

DMF (0.015 M), fluoresceine-5-isothiocyanate (FITC, 3.0 equiv.); diisopropylethylamine (6.0 equiv), 4 h,

then prep. HPLC, 12-25% yield overall. THF = tetrahydrofuran, DMF = dimethylform

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Scheme 1

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Arndt, Eva M. Neuhaus and Hanns HattLian Gelis, Nikolina Jovancevic, Sophie Veitinger, Bhubaneswar Mandal, Hans-Dieter

Functional Characterization of the Odorant Receptor 51E2 in Human Melanocytes

published online May 18, 2016J. Biol. Chem. 

  10.1074/jbc.M116.734517Access the most updated version of this article at doi:

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